1. Field of the Invention
The invention relates to derivative compounds of activated polysaccharides and more particularly relates to water-insoluble, activated polysaccharides coupled to fluorescent deoxyribonucleic acid intercalating agents and to the use of such derivative compounds as fluorescent stains to detect deoxyribonucleic acid and deoxyribonucleic acid fragments.
2. Brief Description of the Prior Art
Prior hereto ethidium bromide has been used as a fluorescent stain to detect the presence of deoxyribonucleic acid and fragments thereof separated by gel electrophoresis techniques. The stain has been used by adding it directly to the buffer used in the electrophoresis apparatus, by soaking the electrophoresis gel after electrophoretic separation of the fragments and by admixing the stain with the gel prior to electrophoresis; see for example Sugden et al, Analytical Biochemistry, 68, 36-46 (1975) and Sharp et al, Biochemistry, 12, No. 16, 3055-9 (1973). Those skilled in the art appreciate that the handling and disposal of free ethidium bromide poses hazards to the operator and others subject to exposure since ethidium bromide has been identified as a potential carcinogen. In general, other fluorescent stains capable of intercalating with deoxyribonucleic acids or fragments thereof pose special problems of handling, i.e.; toxicity, allergenicity and like problems.
The novel compounds of the present invention include a bound-up, but functionally active, fluorescent deoxyribonucleic acid intercalating agent as a fluorescent stain. The agent is chemically bound, i.e.; covalently bonded through chemical groups (such as amine groups) to the polysaccharide component and will not diffuse or wash from the gel as will the prior art physical mixes of gels with such agents. For example, in those compounds binding an ethidium halide as the intercalating agent, the ethidium halide is not diffused or washed from its chemical binding, resulting in a reduction of exposure hazard to the electrophoresis operator and others concerned with disposal of the used agent. The derivative compounds of the invention are further advantageous in that they may be readily included with electrophoresis grade gels such as agarose gels, agarosepolyacrylamide gels and the like by simple admixture therewith. There is an avoidance of separate steps of staining the electrophoretic gel before or after use. This has heretofore been a time consuming procedure which sometimes results in diffusion of the deoxyribonucleic acid bands fractionated on the gel surface. The use of the novel derivative compounds of this invention as stain ingredients in electrophoretic gels also appears to provide better resolution of the fractionation, a particular advantage for example in two dimensional electrophoresis procedures.
The derivative compounds of the invention, when used as ingredients of electrophoretic grade gels as described more fully hereinafter do not appear to reduce the mobility of deoxyribonucleic acids or fragments thereof on the gels during electrophoresis nor do they appear to otherwise interfere with or adversely affect the desired electrophoretic fractionation.